In the clinical diagnosis of bacterial diseases, such as tuberculosis, a sample of sputum or other body fluid obtained from the patient is cultured to test for the presence of the particular bacterium of interest. Unfortunately, this is a relatively time-consuming process, generally requiring several days to produce a definitive result. During this time, a patient suspected of having tuberculosis, for example, must be isolated to prevent further spread of the disease.
The advent of DNA probes, which can identify a specific bacteria by testing for the presence of a unique bacterial DNA sequence in the sample obtained from the patient, has greatly increased the speed and reliability of clinical diagnostic testing. A test for the tuberculosis mycobacterium, for example, can be completed within a matter of hours using DNA probe technology. This allows treatment to begin more quickly and avoids the need for long patient isolation times.
In the use of DNA probes for clinical diagnostic purposes, a nucleic acid amplification reaction is usually carried out in order to multiply the target nucleic acid into many copies or amplicons. Examples of nucleic acid amplification reactions include strand displacement amplification (SDA) and polymerase chain reaction (PCR). Detection of the nucleic acid amplicons can be carried out in several ways, all involving hybridization (binding) between the target DNA and specific probes.
Many common DNA probe detection methods involve the use of fluorescein dyes. One known detection method is fluorescence energy transfer. In this method, a detector probe is labeled both with a fluorescein dye that emits light when excited by an outside source, and with a quencher which suppresses the emission of light from the fluorescein dye in its native state. When DNA amplicons are present, the fluorescein-labeled probe binds to the amplicons, is extended, and allows fluorescence emission to occur. The increase of fluorescence is taken as an indication that the disease-causing bacterium is present in the patient sample.
Several types of optical readers or scanners exist which are capable of exciting fluid samples with light, and then detecting any light that is generated by the fluid samples in response to the excitation. For example, an X-Y plate scanning apparatus, such as the CytoFluor Series 4000 made by PerSeptive Biosystems, is capable of scanning a plurality of fluid samples stored in an array or plate of microwells. The apparatus includes a scanning head for emitting light toward a particular sample, and for detecting light generated from that sample. The apparatus includes first and second optical cables each having first and second ends. The first ends of the optical cables are integrated to form a single Y-shaped "bifurcated" cable. The scanning head includes this end of the bifurcated optical cable. The second end of the first optical cable of the bifurcated cable is configured to receive light from a light emitting device, such as a lamp, and the second end of the second cable of the bifurcated cable is configured to transmit light to a detector, such as a photomultiplier tube.
During operation, the optical head is positioned so that the integrated end of the bifurcated optical fiber is at a suitable position with respect to one of the microwells. The light emitting device is activated to transmit light through the first optical cable of the bifurcated optical cable such that the light is emitted out of the integrated end of the bifurcated optical cable toward the sample well. If fluid sample fluoresces in response to the emitted light, the light produced by the fluorescence is received by the integrated end of the optical fiber and is transmitted through the second optical fiber to the optical detector. The detected light is converted by the optical detector into an electrical signal, the magnitude of which is indicative of the intensity of the detected light. This electrical signal is processed by a computer to determine whether the target DNA is present or absent in the fluid sample based on the magnitude of the electrical signal.
In this type of X-Y plate reader apparatus, the reader head must be repositioned for each well. Accordingly, if the microwell array is a standard microwell array having 12 columns of 8 microwells (96 microwells total), the reader head must move 96 times for the entire microwell array to be read. This excessive movement increases the amount of wear and tear experienced by the apparatus. Furthermore, the control system for controlling the positioning of the head reader must be sophisticated enough to ensure that the integrated end of the optical fiber in the reader head is positioned correctly for each microwell so that the readings are taken at identical locations (e.g., the center) of each microwell. If the integrated end of the optical fiber is not aligned correctly with the microwell, the fluid in the microwell may not receive an adequate amount of excitation light and may therefore not fluoresce properly. Furthermore, any fluorescence that does occur may not be completely detected, because that light may not transmit properly into the integrated end of the bifurcated optical fiber. Accordingly, unless the positioning of the head reader is maintained precise for each individual well, erroneous readings may occur.
Another existing type of apparatus is described in U.S. Pat. No. 5,473,437, to Blumenfeld et al. This apparatus includes a tray having openings for receiving bottles of fluid samples. The tray includes a plurality of optical fibers which each have an end that terminates at a respective opening in the tray. The tray is connected to a wheel, and rotates in conjunction with the rotation of the wheel. The other ends of the optical fibers are disposed circumferentially in succession about the wheel, and a light emitting device is configured to emit light toward the wheel so that as the wheel rotates, the ends of the optical fibers sequentially receive the light being emitted by the light emitting device. That is, when the wheel rotates to a first position, a fiber extending from the wheel to one of the openings becomes aligned with the optical axis of the light emitting device and thus, the emitted light will enter that fiber and be transmitted to the opening. The apparatus further include a light detector having an optical axis aligned with the optical axis of the emitted light. Accordingly, if the sample in the bottle housed in the opening fluoresces due to the excitation light, the light emitted from the sample will transmit through the optical fiber and be detected by the detector. The wheel then continues to rotate to positions where the ends of the other optical fibers become aligned with the optical axis of the light emitter and light detector, and the light emission and detection process is repeated to sample the fluid samples in the bottles housed in the openings associated with those fibers.
As with the CytoFluor X-Y plate reader apparatus described previously, the apparatus described in U.S. Pat. No. 5,473,437 uses a single light emitter and a single light detector to test a plurality of fluid samples. However, instead of using a single bifurcated cable as in the X-Y plate reader apparatus, this apparatus uses a plurality of single optical cables which are individually dedicated to a particular sample. Nevertheless, like the X-Y plate reader apparatus, this apparatus requires mechanical movement between the fluorescent interrogation for each sample. That is, the apparatus is incapable of testing a plurality of samples with only one mechanical motion. Rather, the wheel must rotate to align the optical axis of the light emitter and light detector with each of the optical fibers associated with each of the respective samples. Because the control system used by the apparatus must assume that the appropriate optical fiber is aligned correctly for each sample, the chance of misalignment is significant. Furthermore, this constant movement imposes significant wear and tear on the apparatus.
Another type of optical testing apparatus is described in U.S. Pat. No. 5,518,923, to Berndt et al. That apparatus includes a plurality of light emitter/light detector devices for testing a plurality of fluid samples. The fluid samples are contained in jars which are placed in the openings of a disk-shaped tray. The plurality of light emitter/detector devices are disposed in the radial direction of the tray. Hence, as the tray rotates, the samples in each circular row will pass by their respective light emitter/detector device, which will transmit light into the sample and detect any light that is generated by the sample in response to the emitted light. In theory, this apparatus is capable of testing more than one sample at any given time. However, in order to achieve this multiple sample testing ability, the system must employ a plurality of light detectors and a plurality of light emitters. These additional components greatly increase the cost of the system. For example, photomultiplier tubes, which are generally quite expensive, are often used as light detector units in devices of this type. Hence, the cost of the unit is generally increased if more than one photomultiplier tube is used. However, it is desirable to use as few photomultiplier tubes as possible to maintain a competitive price for the apparatus. However, devices which employ a single detector (e.g., photomultiplier tube) are incapable of testing a plurality of samples without some type of mechanical motion for each test. Therefore, this multiple detection apparatus is somewhat impractical from a cost standpoint.
A detector apparatus is also described in U.S. Pat. No. 4,343,991, to Fujiwara et al. This apparatus employs a single light detector and a plurality of light emitting devices to read a sample on a sample carrier, which is a substantially transparent medium. In this apparatus, the plurality of light emitting devices transmit light through corresponding optical fibers. The light emitted by the optical fibers passes through the carrier and is received by corresponding optical fibers on the opposite side of the carrier. The receiving fibers terminate at a single light detector and the light emitters are operated to emit light at different times. Hence, light from only one of the emitters passes through the carrier at any given time and is detected by the detector, which outputs a signal proportional to the intensity of the detected light. Therefore, a single detector can be used to detect light from a plurality of light emitting devices. When the light passes through a portion of the carrier that includes a sample, the intensity of the light is decreased because some of the light is absorbed by the sample. The amount by which the light intensity is reduced is proportional to the concentration of the sample material in the sample. Because the signal output by the detector is proportional to the intensity of the detected light, the sample concentration can thus be determined based on the output signal.
Although the apparatus described in U.S. Pat. No. 4,343,991 eliminates the need for a mechanical movement for each sampling, the arrangement of the optical fibers on opposite sides of the carrier requires that the fibers be precisely aligned so that the light emitted by an optical fiber is detected properly by its corresponding optical fiber coupled to the light detector. The apparatus is therefore easily susceptible to erroneous readings if the light emitting optical fibers and light detecting optical fibers are not perfectly aligned. Furthermore, the light detecting fibers are not arranged to detect luminescence of the samples, but rather, the intensity of the light passing through the samples. In addition, since the light emitting fibers and light detecting fibers are on opposite sides of the carrier, enough space must be allocated in the apparatus to accommodate fibers on both sides of the area through which the carrier is conveyed, thus increasing the overall size of the apparatus.
Accordingly, a continuing need exists for an optical testing apparatus employing a single detector, such as a photomultiplier tube, which is capable of accurately performing a plurality of tests on a plurality of liquid samples without requiring a mechanical movement for each test.